This proposal seeks to further develop and utilize, in high throughput molecular screening applications, a novel continuous spectroscopic assay for DNA topoisomerases. These enzymes are established targets for anticancer, antibacterial and antiviral drug therapies, and this robust assay now allows for rapid screening of large chemical libraries for new drug candidates for these targets. The assay is especially robust and economical for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. The utility of the assay has been validated, by screening a 2000 member small molecule library from which several novel lead compounds have been identified that specifically target a poxvirus family topoisomerase.